G-CSF (granulocyte-colony stimulating factor) is a hematopoietic cytokine, released mainly by mononuclear cells and fibroblasts, that stimulates the proliferation and differentiation of precursor cells of the granulocyte lineage and the activation of functionally mature neutrophils. Due to said characteristics, G-CSF has come to be used in different medical fields, like for example in the reconstitution of normal blood cell populations subsequent to chemotherapy or irradiation or for stimulating the immune response to infectious pathogens. Thus in the clinics, G-CSF is mainly employed in anti-tumor therapy, particularly in the treatment of neutropenia as a consequence of chemotherapy, and is furthermore used in bone marrow transplantations and in the treatment of infectious diseases. The first commercially available G-CSF preparation based on recombinant G-CSF was produced and distributed by Amgen under the trade name Neupogen®.
Human G-CSF in its naturally occurring form is a glycoprotein having a molecular weight of about 20,000 Dalton and five cysteine residues. Four of these residues form two intramolecular disulfide bridges, which are of essential importance for the activity of the protein. Recombinant forms of G-CSF are mainly used for producing pharmaceuticals, which can for example be obtained by means of expression in mammalian cells like CHO (Chinese Hamster Ovary) cells or in prokaryotic cells like E. coli. When recombinant proteins are expressed in prokaryotes the proteins are often produced within the host cell in the form of at least partially inactive, insoluble aggregates (refractile bodies, inclusion bodies IB). Before such proteins can be used they have to be converted into their active form.
The formation of said inclusion bodies leads to the necessity of solubilizing and renaturing the proteins subsequent to isolation of the inclusion bodies by means of centrifugation at moderate speed with the aid of suitable means in order to maintain their active configuration.
Processes for the renaturation of recombinant proteins derived from inclusion bodies are generally known and described for example in EP 0 114 506, WO 84/03711, U.S. Pat. No. 4,530,787 and EP 0 241 022. In addition, general techniques relating to solubilization and renaturing of denatured proteins have been described in EP 0 512 097, EP 0 364 926, EP 0 219 874 and WO 01/87925 and can furthermore be taken from scientific literature and standard works on protein chemistry.
EP 0 500 108 describes a method for activating human recombinant G-CSF in an inactive form from inclusion bodies using a reduced glutathione (GSH) and oxidized glutathione (GSSH) redox shuffling system and analysis of reactivation kinetics for G-CSF under certain conditions. However, a downstream purification process is not disclosed.
In EP 0 719 860, G-CSF containing inclusion bodies were solubilized with N-Lauroylsarcosine (sarcosyl) and subsequently refolding was achieved by air-oxidation using copper sulfate. The disadvantages of this method are side reactions, e.g., formation of superoxide radicals on amino acid side chains. Furthermore, the refolding process is time consuming and it is difficult to obtain standardized refolding parameters. Finally, removal of the denaturant including a chromatographic step leads to a loss of about 20% of total protein yield. The G-CSF obtained is subsequently purified by an anion exchange chromatography and a cation exchange chromatography.
EP 1 630 173 and EP 1 837 346 describe methods of obtaining human recombinant G-CSF from inclusion bodies using a reduced glutathione (GSH) and oxidized glutathione (GSSH) redox shuffling system, wherein the refolding step is performed at low temperatures for more than half a day. Therefore, at an industrial scale this process is energy and thus cost consuming due to cooling large volumes of protein solution over many hours. The resulting G-CSF is subsequently purified by cation exchange chromatography.
In WO2007/009950 the method for purification of G-CSF taught in EP 1 630 173 is further specified with respect to the chromatographic steps in that cation exchange chromatography and hydrophobic interaction chromatography are consecutively performed without any intermediate step in between. In particular, the chromatographic purification procedure comprises a sequence of two cation exchange chromatography steps conducted before and after the hydrophobic interaction chromatography, respectively.
However, while means and methods for providing purified G-CSF at therapeutic grade were known in the prior art, processes available to date for obtaining G-CSF from inclusion bodies, in particular at a commercial scale are generally time-, labor- and cost-consuming. This technical problem is solved by the embodiments as characterized in the claims and described further below. In addition, as described further below, these embodiments provide for separation of a conformational G-CSF isoform and, therefore, a highly homogeneous G-CSF preparation.